Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.

Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.

Cell Subtypes Within the Liver Microenvironment Differentially Interact with Lipid Nanoparticles.

INTRODUCTION: Lipid nanoparticles (LNPs) tend to accumulate in the liver due to physiological factors. Whereas the biological mechanisms that promote LNP delivery to hepatocytes have been reported, the mechanisms that promote delivery to other cell types within the liver microenvironment are poorly understood. Single cell profiling studies have recently identified subsets of Kupffer cells and hepatic endothelial cells with distinct gene expression patterns and biological phenotypes; we hypothesized these subtypes would differentially interact with nanoparticles. METHODS: To test the hypothesis, we quantified nucleic acid (i) biodistribution and (ii) functional mRNA delivery within the liver microenvironment using two clinically relevant LNPs in vivo. RESULTS: We found that these LNPs distribute nucleic acids distribute to Kupffer cells and liver endothelial cells as efficiently as they distribute to hepatocytes, yet result in more functional mRNA delivery to endothelial cells. Additionally, we found these LNPs differentially accumulate in Kupffer and endothelial cell subsets. CONCLUSIONS: These data suggest subsets of liver microenvironmental cells can differentially interact with nanoparticles in vivo, thereby altering LNP delivery. More generally, the data suggest that nucleic acid biodistribution is not sufficient to predict functional nucleic acid delivery in vivo.

Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands.

T cells help regulate immunity, which makes them an important target for RNA therapies. While nanoparticles carrying RNA have been directed to T cells in vivo using protein- and aptamer-based targeting ligands, systemic delivery to T cells without targeting ligands remains challenging. Given that T cells endocytose lipoprotein particles and enveloped viruses, two natural systems with structures that can be similar to lipid nanoparticles (LNPs), it is hypothesized that LNPs devoid of targeting ligands can deliver RNA to T cells in vivo. To test this hypothesis, the delivery of siRNA to 9 cell types in vivo by 168 nanoparticles using a novel siGFP-based barcoding system and bioinformatics is quantified. It is found that nanomaterials containing conformationally constrained lipids form stable LNPs, herein named constrained lipid nanoparticles (cLNPs). cLNPs deliver siRNA and sgRNA to T cells at doses as low as 0.5 mg kg-1 and, unlike previously reported LNPs, do not preferentially target hepatocytes. Delivery occurs via a chemical composition-dependent, size-independent mechanism. These data suggest that the degree to which lipids are constrained alters nanoparticle targeting, and also suggest that natural lipid trafficking pathways can promote T cell delivery, offering an alternative to active targeting approaches.

Nanoparticles Containing Oxidized Cholesterol Deliver mRNA to the Liver Microenvironment at Clinically Relevant Doses.

Using mRNA to produce therapeutic proteins is a promising approach to treat genetic diseases. However, systemically delivering mRNA to cell types besides hepatocytes remains challenging. Fast identification of nanoparticle delivery (FIND) is a DNA barcode-based system designed to measure how over 100 lipid nanoparticles (LNPs) deliver mRNA that functions in the cytoplasm of target cells in a single mouse. By using FIND to quantify how 75 chemically distinct LNPs delivered mRNA to 28 cell types in vivo, it is found that an LNP formulated with oxidized cholesterol and no targeting ligand delivers Cre mRNA, which edits DNA in hepatic endothelial cells and Kupffer cells at 0.05 mg kg-1 . Notably, the LNP targets liver microenvironmental cells fivefold more potently than hepatocytes. The structure of the oxidized cholesterols added to the LNP is systematically varied to show that the position of the oxidative modification may be important; cholesterols modified on the hydrocarbon tail associated with sterol ring D tend to outperform cholesterols modified on sterol ring B. These data suggest that LNPs formulated with modified cholesterols can deliver gene-editing mRNA to the liver microenvironment at clinically relevant doses.

Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution.

Bone marrow endothelial cells (BMECs) regulate their microenvironment, which includes hematopoietic stem cells. This makes BMECs an important target cell type for siRNA or gene editing (e.g., CRISPR) therapies. However, siRNA and sgRNA have not been delivered to BMECs using systemically administered nanoparticles. Given that in vitro nanoparticle screens have not identified nanoparticles with BMEC tropism, we developed a system to quantify how >100 different nanoparticles deliver siRNA in a single mouse. This is the first barcoding system capable of quantifying functional cytosolic siRNA delivery (where the siRNA drug is active), distinguishing it from in vivo screens that quantify biodistribution (where the siRNA drug went). Combining this approach with bioinformatics, we performed in vivo directed evolution, and identified BM1, a lipid nanoparticle (LNP) that delivers siRNA and sgRNA to BMECs. Interestingly, chemical analysis revealed BMEC tropism was not related to LNP size; tropism changed with the structure of poly(ethylene glycol), as well as the presence of cholesterol. These results suggest that significant changes to vascular targeting can be imparted to a LNP by making simple changes to its chemical composition, rather than using active targeting ligands. BM1 is the first nanoparticle to efficiently deliver siRNA and sgRNA to BMECs in vivo, demonstrating that this functional in vivo screen can identify nanoparticles with novel tropism in vivo. More generally, in vivo screening may help reveal the complex relationship between nanoparticle structure and tropism, thereby helping scientists understand how simple chemical changes control nanoparticle targeting.